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J. Biol. Chem., Vol. 275, Issue 45, 35320-35327, November 10, 2000
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From The Institute of Medical Biochemistry, Department of Molecular
Genetics, Biocenter and University of Vienna, Vienna A-1030,
Austria
Received for publication, June 12, 2000, and in revised form, August 16, 2000
Development of the follicle in egg-laying species
such as the chicken is regulated by systemic factors as well as by the
highly orchestrated interplay of differentially expressed genes within this organ. Differential mRNA display analysis of defined phases of
follicle development resulted in the characterization of coagulation factor XIIIA. It is expressed and produced by cells of the theca externa in a highly regulated manner during distinct growth phases of
the follicle. Transcripts for factor XIIIA are already detectable at
the beginning of follicle development and peak at the end of phase 2. Protein levels, however, still increase during phase 3, peak shortly
after ovulation, and persist until the postovulatory tissue is
completely resorbed. Factor XIIIA is secreted as a monomer into the
extracellular matrix of the theca externa and is not associated with
factor XIIIB as is the case in plasma. Our data suggest that, due to
its transglutaminase activity, factor XIIIA stabilizes the follicular
wall by cross-linking matrix components. Thus, coagulation factor XIIIA
might play a key role in coping with the massive mechanical stress
exerted by the large amount of yolk accumulating during the rapid
growth phase of the oocyte.
Reproduction in the mature hen depends on the coordinate
differentiation and growth of oocytes. In the case of the domesticated chicken (Gallus gallus domesticus), fully developed oocytes
are laid as eggs every 25 h. The oocytes develop in follicles,
highly specialized structures of the ovary consisting of the oocyte
proper in the center of the follicle, surrounded by concentric layers of cells, acellular material, and structures, including the
perivitelline membrane (the equivalent of the zona pellucida in
mammals), granulosa cells, a basement membrane, and the thecae interna
and externa (1). Development of follicles can be divided into three
major phases (for review see Ref. 2). Phase 1 is characterized by a
very slow growth of follicles lasting for several months. At the end of
this period, numerous follicles have reached a diameter of 2-3 mm; the
oocytes within these follicles do not contain significant amounts of
bona fide yolk (these follicles are referred to herein as
small white follicles). During phase 2, some of these follicles develop further and reach a diameter of approximately 6 mm after 60 days. Due to yolk deposition into the oocyte, these follicles acquire a
yellow appearance (here termed small yellow follicles). Finally, single
follicles are selected from the pool of small yellow follicles every
25 h (synchronous with the ovulation cycle) and enter the rapid
growth phase, which leads to mature follicles with a diameter of
approximately 35 mm within 7 days. The fully developed oocyte is
expelled from the follicle by ovulation and enters the oviduct, where
egg formation starts. After ovulation, the remaining structure of the
follicle, termed postovulatory follicle
(POF),1 consists of the
granulosa cells, basement membrane, and theca layers. The POF
collapses, but stays metabolically active for up to 6 days before
involution and resorption (2). This developmental scheme establishes a
hierarchy of follicles present at any given time in the ovary of a
mature hen. Typically, the ovary contains five to eight prominent
preovulatory follicles in the rapid 7-day growth phase. These follicles
are numbered, from F1, the largest one, which will ovulate next, to the
smallest distinguishable ones, usually F5 through F8.
Despite our detailed knowledge about vitellogenesis (for review see
Refs. 3 and 4) and steroid production in the follicle (for review see
Ref. 2), very little is known about the orchestration of these events
at the molecular level. Especially, mechanisms responsible for
establishing the follicular hierarchy are completely unknown.
Additionally, another interesting point is the cellular and acellular
architecture of the follicular wall. Although there is considerable
knowledge about the morphology of this structure, very little is known
about molecular and genetic aspects of cell differentiation and
production of extracellular components during development of the
follicle and its remodeling in preparation for and following ovulation.
We are approaching these questions from two angles. First, we have
recently begun to study and characterize the production of the
acellular structures within the follicles, perivitelline membrane, and
the basement membrane. We found, e.g. that one of the zona
pellucida proteins, ZPC, is synthesized by granulosa cells and
deposited in polarized fashion into the space between granulosa cells
and the oocyte (5). ZP1, another constituent of the perivitelline
membrane, however, is synthesized in the liver, transported via the
bloodstream to the ovary, and becomes deposited in the perivitelline
membrane (6). Because follicle development takes place simultaneously,
but non-synchronously in many different follicles, we have to assume
that the program guiding these complex mechanisms is not controlled by
systemic factors, but rather within the follicle itself. We expect a
highly orchestrated interplay of differentially expressed genes within different cells, dependent on growth phases of the follicle. Thus, as
part of our second approach we have started to screen for such genes
using the differential mRNA display methodology. Here we report
results obtained in such a screen for genes expressed at various levels
during different phases of follicle development. We show that the avian
homologue of human coagulation factor XIIIA is expressed and secreted
by theca cells and propose that it stabilizes the follicular wall by
cross-linking matrix components during the last growth spurt of the
oocyte, in preparation for ovulation of the follicle, for its
subsequent transition into the postovulatory follicle and resorption.
Differential Display PCR--
Total RNA from chicken follicles
was prepared with TRI reagent (Molecular Research Center, Inc.).
Poly(A)+ RNA was prepared with the Oligotex mRNA kit
(Qiagen). 1 µg of poly(A)+ RNA was mixed with 50 pmol of
oligo-dT primer, incubated at 70 °C for 10 min, and chilled on ice.
First strand cDNA was synthesized in 20 µl of buffer containing
2.5 µM of each dNTP, 50 mM Tris-HCl (pH 8.3),
40 mM KCl, 6 mM MgCl2, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 200 units of Superscript II reverse transcriptase (Life Technologies, Inc.)
at 42 °C for 50 min. The reaction mixture (20 µl) for differential
display (DD)-PCR contained 1 µl of diluted cDNA (1:15), 1 µM arbitrary primer (5'-TACAACGAGG-3'), 1 µM anchored primer (5'-T11AC-3'), 2.5 µM of each dNTP, 5 µCi of [ Isolation of Chicken Factor XIIIA cDNA--
DD-PCR clone 12 was used as a probe for screening a chicken follicle cDNA library
(7) using standard conditions. Positive clones were subcloned into
pBluescript II SK (Stratagene) and sequenced. To obtain the 5'-end of
the coding sequence, 5'-rapid amplification of cDNA ends (RACE) was
employed using the Marathon cDNA amplification kit
(CLONTECH). The primers used were
5'-TCCTTCTCGTCATCCAGGTACACAGC-3' and
5'-TCAGCACATCACCTATGAGGATCGG-3'.
Expression of Chicken FXIIIA--
The full-length cDNA of
chicken FXIIIA was cloned into the XbaI and SmaI
sites of pCIneo (Promega) and expressed in the human embryonic kidney
cell line 293. Transfection of the cells was performed using Lipofectin
reagent (Life Technologies, Inc.). Stable transformants were selected
by the addition of 500 mg/liter G418 to the medium (Dulbecco's
modified Eagle's medium (Life Technologies, Inc.), 10% fetal calf
serum, 584 mg/liter glutamine). Cells expressing factor XIIIA were
washed three times with 2 ml each of phosphate-buffered saline (PBS),
scraped from the dishes with a rubber policeman, and centrifuged at
2000 × g for 5 min, and the cell pellet was solubilized by addition of 150 µl of buffer/dish (200 mM
Tris maleate, pH 6.5, 2 mM CaCl2, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 µM
leupeptin, and 1.4% Triton X-100). The cell extracts were centrifuged
at 300,000 × g for 40 min at 4 °C and the pellet discarded.
Purification of Glutathione S-Transferase Fusion Proteins and
Antibody Production--
cDNA fragments encoding amino acid
residues 1-45 (corresponding to the activation peptide) and amino acid
residues 674-735 (corresponding to the carboxyl terminus) of chicken
factor XIIIA were cloned into pGEX-5X-1 (Amersham Pharmacia Biotech).
Isolation of the glutathione S-transferase fusion proteins
in DH5 Immunoprecipitation and Western Blotting--
For
immunoprecipitation, 1 ml of conditioned medium (from 293 cells
expressing FXIIIA and mock-transfected cells) was incubated for 24 h at 4 °C with 5 µl of antiserum (Ac16) or preimmune serum and with 4 µg of Protein A-Sepharose (Amersham Pharmacia Biotech) in
10 mM Tris/HCl, 150 mM NaCl, 2 mM
EDTA, pH 7.4. Then the Sepharose beads were washed five times with
ice-cold PBS. Proteins were eluted from the beads with Laemmli sample
buffer containing Immunofluorescence--
Follicles (6 mm) from an adult hen were
dissected in ice-cold PBS, embedded in freezing agent (Microm,
Austria) and immediately frozen. Cryostat sections of 12-µm thickness
were prepared, transferred to Superfrost-Plus slides (Menzel) and
stored at In Situ FXIIIA Activity Assay--
Transglutaminase activity was
detected as described previously (11). Briefly, slides prepared as
described above were washed with PBS, preincubated in 100 mM Tris-HCl, pH 8, 1% bovine serum albumin, 10 mM CaCl2 or 10 mM EDTA for 1 h
at room temperature, and incubated with fluorescein-labeled cadaverine
(Molecular Probes) in blocking buffer for 1 h at room temperature.
After washing, microscopy was performed on a Zeiss microscope (Axiovert 135).
In a first step to identify genes involved in the progression of
follicle development, mRNAs isolated from three distinct pools of
differently staged follicles (small white, <2 mm; large white, 2-4
mm; small yellow, 4-8 mm), covering phases 1 and 2 of follicle
development, were subjected to differential mRNA display analysis
(12). We have isolated several fragments derived from genes that
appeared up- or down-regulated as judged from the intensities on the
gel, e.g. fragments 12 and 13 displayed in Fig.
1A. However, only six of these
fragments (derived from mRNAs displayed using the given primer
combination) exhibited differential expression, as evaluated by
Northern blotting (Fig. 1, B and C). To obtain sequence information from the coding regions corresponding to the
selected fragments, these fragments were used to screen a chicken
follicle cDNA library (7). BLAST searches against available data
bases demonstrated that only one of the clones (number 12) was
identical with a known chicken gene; it codes for a chicken chondrocyte
transglutaminase (13, 14). Thus, we focused our attention on clone 12, and the sequence of the corresponding full-length transcript with a
length of 4.5 kilobase pairs was obtained by 5'-RACE. The sequence
defined an open reading frame of 2208 base pairs (GenBankTM accession
number AJ278103). Sequence alignments (Fig.
2) clearly identified the obtained
cDNA as the chicken homologue of human factor XIIIA (15). The ATG
serving as the translational start site was assigned based on the
following considerations: First, it is located in a sequence context
fulfilling the rules for a translation initiation site (16), and
second, the corresponding chicken protein starts with the same sequence
motif as human factor XIIIA (Fig. 2). The overall identity at the amino
acid level between chicken and human factor XIIIA is 65%, displaying
100% identity around the active site cysteine, which catalyzes the
acyl transfer reaction common to all transglutaminases (for review see
Ref. 17).
Mammalian plasma factor XIIIA is part of a large tetrameric complex
consisting of two molecules of XIIIA and two molecules of XIIIB. The
cellular form of factor XIIIA, however, is a homodimer consisting of
two A subunits only. An interesting functional feature of human factor
XIIIA is a thrombin cleavage site between Arg-37 and Glu-38 (see
Fig. 2). Cleavage of FXIIIA by thrombin at this site leads to
liberation of the amino-terminal so-called activation peptide and
subsequently to the dissociation of the A and B subunits. Thus, in the
absence of the inhibitory B subunits, the thrombin-modified A subunits
assume a Ca2+-dependent active conformation
catalyzing the cross-linking of fibrinogen to insoluble fibrin.
To characterize the corresponding chicken protein further, we produced
two antibodies directed against the putative activation peptide at the
amino terminus (Ac16), and against the extreme carboxyl terminus of
chicken XIIIA (A3), respectively. Using these antibodies in combination
with a commercially available antibody against human XIIIA, we first
characterized the plasma form of chicken XIIIA (Fig.
3A). Under non-reducing
conditions, Ac16 recognizes all three forms of chicken factor XIIIA in
plasma, i.e. the heterotetramer (2A2B), the homodimer (2A),
and the monomer (A), respectively (lane 1). The occurrence
of both the homodimer (2A) and the unclipped monomer (A) in plasma is
due to the inherent instability of the tetrameric complex during
electrophoresis. Under the same conditions, A3 and the commercial
anti-human-XIIIA antibody recognize the heterotetramer in plasma
(lanes 3 and 5). Independently of the enzyme's
activation status, A3 reacts with the dimer as demonstrated by
comparing plasma versus serum samples electrophoretically
separated under non-reducing conditions (lanes 3 and
4). A3 only weakly recognizes the monomer of chicken XIIIA
under the conditions tested (the monomer becomes visible only after
prolonged exposure, lane 3). Removal of the activation
peptide and dissociation of the A and B subunits was demonstrated by
the absence in serum of any detectable heterotetramer using A3 and
anti-human XIIIA (lanes 4 and 6). In addition,
under reducing conditions, all of the XIIIA present in plasma
(i.e. not activated) migrates as monomer and is strongly
recognized by Ac16 (lane 7). Upon activation, in serum, hardly any reactive band is visible under reducing conditions, indicating that the activation peptide is removed (lane 8).
Upon prolonged exposure of the blot, however, a faint band with a size similar to that of the monomer becomes apparent; most likely it represents a small residual amount of non-activated XIIIA (also visible
as dimer under non-reducing conditions in lane 2). Thus, activation of chicken factor XIII, as it occurs during blood clotting, obviously follows a similar process as described for mammals (17).
Chicken Coagulation Factor XIIIA Is Produced by the Theca Externa
and Stabilizes the Ovarian Follicular Wall*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-35S]dATP,
20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.25 mM MgCl2, 2.5 units of Taq
polymerase (Life Technologies, Inc.). After an incubation of 4 min at
94 °C, the mixture was subjected to 40 low stringency cycles
(94 °C for 30 s, 40 °C for 2 min, 72 °C for 30 s)
and a final extension at 72 °C for 30 s. 5 µl of the
amplified cDNA fragments was separated on a 8% polyacrylamide 8 M urea gel. Bands were visualized by exposing the dried gel
to x-ray film (Kodak X-Omat). Gel pieces containing the bands of
interest were excised from the gel and rehydrated in 150 µl of water
by boiling for 30 min. Then the DNA was precipitated with ethanol and
after dissolving in water used for reamplification as described for the
original DD-PCR, except for the use of [-35S]dATP.
Reamplified DNA fragments were cloned into pCR2.1 (Invitrogen) and
sequenced by the dideoxy chain termination method using the Sequenase
Version 2.0 DNA sequencing kit (United States Biochemical). Sequence data obtained were subjected to homology search at nucleotide levels using the BLAST search of the National Institutes of Health.
cells (8) and production of the respective polyclonal
antibodies (9) were carried out as described in the indicated
references. The antisera were designated Ac16 (against the activation
peptide) and A3 (against the carboxyl terminus).
-mercaptoethanol (5%, v/v) and separated by
SDS-polyacrylamide gel electrophoresis. Presence of chicken factor
XIIIA in the immunoprecipitates was tested by Western blotting as
described (10).
70 °C. Slides were washed in PBS, incubated for 24 h with the respective primary antibody (Ac16; anti--collagen
I, Sigma; anti-chicken-fibronectin, Chemicon Int.; anti-human-factor
XIIIA, Calbiochem), washed, and incubated with a fluorescein
isothiocyanate-conjugated anti-rabbit IgG (Sigma). After a final wash,
microscopy was performed on a Zeiss microscope (Axiovert 135).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 1.
Differential display of mRNAs from
different developmental stages of chicken follicles.
Poly(A)+ RNA from three different stages of follicle
development were subjected to differential mRNA display analysis as
described under "Experimental Procedures." A partial autoradiogram
showing two selected mRNAs (#12, #13) is
presented in A. B and C demonstrate
the expression pattern of #12 and #13 as assessed by Northern blotting.
Relative intensities of the bands normalized to the amount of 18 S rRNA
are shown in the corresponding diagrams underneath the Northern blots.
sw, small white follicles; sy, small yellow
follicles; lw, large white follicles.
View larger version (101K):
[in a new window]
Fig. 2.
Sequence comparison of chicken and human
factor XIIIA. Numbering of the amino acid sequences start at the
methionine residue corresponding to the initiation codon. Gaps ( )
have been introduced to optimize alignment. Identical residues in both
proteins are boxed. The filled arrowhead marks
the site where thrombin cleaves. The arrow marks the active
site cysteine. The Ca2+-binding site is
underlined.
View larger version (26K):
[in a new window]
Fig. 3.
Western blot analysis of chicken factor XIIIA
in plasma and follicles. Plasma (P) and serum
(S) samples (0.5 µl) (panel A) and chicken
follicle extracts (50 µg of protein) (panel B) were
electrophoretically separated under reducing (r) or
non-reducing (n) conditions and analyzed by Western blotting
using the indicated antibodies as described under "Experimental
Procedures." The primary antibody was visualized with
HRP-goat-anti-rabbit IgG (1:10,000) and a chemiluminescence system.
Exposure time was 1 min for A and 20 s for
B. The positions of the heterotetramer, homodimer, and
monomer of factor XIIIA are indicated.
Having characterized the circulating forms of chicken factor XIIIA, we studied the protein produced by chicken follicles. Under both reducing and non-reducing conditions, the only band detected by Ac16 is the monomer, indicating that XIIIA is not associated with the B subunit and that follicular XIIIA does not form a dimer as it does in the circulation (Fig. 3B, lane 1). This observation is substantiated by using A3, which reacts strongly with the dimer (2A) and the heterodimer of XIIIA (2A2B), but only weakly with the monomer (A), as demonstrated on plasma and serum samples. In the follicle this antibody visualizes only the monomer under the conditions applied (Fig. 3B, lanes 3 and 4).
Human factor XIIIA does not contain a hydrophobic leader sequence and
thus belongs to a group of proteins which are secreted from cells by an
"unusual secretory pathway" (for review see Ref. 18). Chicken
factor XIIIA also does not contain a hydrophobic leader sequence (Fig.
2). To test whether chicken factor XIIIA can be secreted from cells, we
expressed the full-length cDNA in 293 cells and tested for the
expressed protein in cell extracts and the medium. As seen in Fig.
4A, transfected 293 cells
express large amounts of monomeric chicken factor XIIIA (lane
2). Again, antibody A3 does not detect a band corresponding to the
homodimer seen under the same conditions in the plasma (Fig. 4,
lanes 4 and 6). Furthermore, significant amounts
of the non-activated monomeric protein are indeed secreted into the
medium, as demonstrated by immunoprecipitation experiments using Ac16
(Fig. 4B, lanes 1-5).
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To evaluate the expression pattern of XIIIA in chicken tissues, we examined all major organs (follicle, eye, intestine, liver, kidney, muscle, brain, heart, lung, skin, adrenal, and spleen) and relevant cell lines by Northern blot analysis (data not shown). The only site in female chicken expressing detectable transcripts for factor XIIIA is the follicle and, of the cell lines tested, HD11 cells, a chicken macrophage cell line (19).
From these experiments we conclude that in the chicken circulating factor XIIIA is highly homologous to human factor XIIIA and that it is associated into a tetrameric complex constituting plasma factor XIII in a similar way as described for the mammalian system. Expression analysis did not reveal where the circulating protein is produced. This situation is reminiscent of that in humans where the question about the site of synthesis of plasma-borne XIIIA is still not resolved (17). However, the follicle produces the monomeric form of factor XIIIA, and its regulated expression during follicle development suggests that it plays a role in this complex process.
To study this unexpected finding, we examined in close detail the
expression pattern of follicular XIIIA during the entire sequence of
follicle development, starting at phase 1 to the complete involution of
the postovulatory follicle. Such an experiment is hampered by the fact
that follicles taken from the last growth phase (F8-F1) contain up to
15 g of yolk. RNA preparation from such follicles is very
inefficient, and data obtained cannot be quantified appropriately.
Thus, we have included follicles from phase 1 up to the smallest
follicles at the beginning of phase 3 (F8-F6), and representative
postovulatory follicles covering the time period from after ovulation
until almost complete resorption. Detailed evaluation on follicles
during the last rapid growth phase will be presented in a separate
experiment below. As shown in Fig. 5,
transcripts for factor XIIIA are already present at the very beginning
of follicle development, and the amount significantly increases until
follicles reach the end of phase 2 (5-8 mm). This result confirms the
initial finding from the differential mRNA display approach that
led to the characterization of factor XIIIA in follicles. Apparently,
mRNA levels decrease at the beginning of the rapid growth phase
(approximately 8 mm) and further decrease after ovulation (Fig. 5,
top). However, protein levels seem to increase during the
last phase of follicle development, reach the highest levels shortly
after ovulation, and stay high until the postovulatory tissue is
completely resorbed (Fig. 5, bottom). This observation
suggests that factor XIIIA is not catabolized during follicle
development, but rather becomes deposited in the extracellular matrix
of the follicle wall. To further evaluate this question and to pinpoint
the site of synthesis of factor XIIIA in the follicle, we turned to the
largest follicles (F4-F1) of the final growth phase. Despite problems
to isolate RNA from follicles of this size, they can be dissected to
separate and prepare the theca and granulosa cell layers free of most
of the yolk (20). RNA and protein extracts were prepared from these tissues and subjected to Northern and Western blot analysis. The most
striking result from this experiment was that expression of factor
XIIIA in the follicle is restricted to the theca layer and virtually
absent from the granulosa cell layer (Fig.
6A). Immunohistochemical
analysis of the follicles showed that factor XIIIA is present in the
theca externa and in the basement membrane but not in theca interna
(Fig. 6B, Panels 1 and 2).
Laminated staining of the theca externa, which is similar to that seen
with anti-collagen-1 antibodies (Fig. 6B, Panel
3) and anti-fibronectin antibodies (Fig. 6B,
Panel 4), together with the fact that granulosa cells do not express the protein, suggest that factor XIIIA is secreted
from theca cells and deposited in the acellular matrix of the theca
externa and possibly in the basement membrane.
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, basement membrane; To evaluate whether factor XIIIA is enzymatically active in the matrix
of the follicle wall, an activity assay based on fluorescein-labeled cadaverine as substrate was performed on thin sections of the follicle.
Background labeling of the sections was controlled by using EDTA (Fig.
7, A* and B*),
which completely blocks the transglutaminase reaction. As seen in Fig.
7A, the distribution of the transglutaminase activity
visualized by incorporation of the labeled substrate is identical with
the distribution of the immunoreactive enzyme seen in Fig.
6B (Panels 1 and 2). Furthermore, the
activity pattern is maintained in the early postovulatory follicle
(Fig. 7B), when the highest amount of factor XIIIA is
present in the follicle (Fig. 5). Note that at this stage the
perivitelline membrane is lost (together with the ovulated oocyte) and
the granulosa cell layer starts to disintegrate, whereas the theca
externa reaches its largest extension.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Differential mRNA display screening for genes expressed during specific stages of chicken follicle development resulted in the identification of chicken factor XIIIA. The transglutaminase is expressed by cells of the theca externa and deposited in certain areas of the follicular wall. Usually, factor XIIIA is part of a large tetrameric complex consisting of two molecules of XIIIA and two molecules of XIIIB constituting plasma coagulation factor XIII. Upon activation by thrombin, factor XIII catalyzes the last step of the blood coagulation pathway by cross-linking soluble fibrin monomers to insoluble fibrin, which forms and stabilizes the final clot. Besides its abundant presence in plasma, factor XIIIA can also be found in certain cells like platelets (21), monocytes/macrophages (22), and some mononuclear cells in the skin (23). The cellular form of factor XIII, however, is a homodimer consisting of two A subunits only. Although the presence of factor XIIIA in these cells has been known for many years, the function of this form is still unclear. Recent reports (13, 14, 24) suggest that plasma factor XIIIA has specialized functions in certain other tissues and cells independent of its function in the blood. It seems to be associated with peripheral nervous tissue and may play a role in the regeneration of this tissue after injury (24). These authors present circumstantial evidence that XIIIA is at least partially synthesized by macrophages present in the nervous tissue. Another interesting aspect of a "non-plasma" form of XIIIA is directly related to the results presented here. Subtractive hybridization to isolate genes involved in hypertrophy of chondrocytes in the chicken lead to the identification of plasma factor XIIIA (13, 14). During chondrocyte hypertrophy, factor XIIIA becomes up-regulated and externalized into the extracellular matrix, where it may serve to stabilize the matrix, which eventually undergoes calcification and the formation of bone structure. In the rat, however, expression of a tissue transglutaminase was described in terminally differentiating chondrocytes (25, 26). Whether factor XIIIA expression in chondrocytes is specific for chicken and whether both transglutaminases serve similar or different roles remain to be established. In any case, factor XIIIA deposited in hypertrophic cartilage in the chicken is not derived from the plasma pool but is directly synthesized by the chondrocytes (14).
What is the function of factor XIIIA in the developing chicken follicle? The answer to this question may lie in the architecture of this continuously changing organ. The structural integrity of the follicle, which reaches a diameter of up to 3 cm and holds a yolk volume of approximately 15 ml at its maximal size before ovulation, is supported mainly by the mechanical strength of the theca externa. This layer is predominantly composed of sheets of fibroblast-like cells embedded in sheets of collagen fibrils (1) and fibronectin (27), giving this layer its stratified appearance (see Fig. 6B). The theca interna, however, contains many capillaries and cell clusters, which, due to their lipid granule content and rounded appearance, have been called thecal gland cells (28). These cells have been reported to be the main site of the production of progesterone and testosterone (29) and of alkaline phosphatase in the follicle (30). In sharp contrast to the theca externa, intercellular spaces of the interna are less well structured and contain only a few scattered collagen fibrils. Very little is known about the structure, composition, and function of the basement membrane that delineates the theca from the granulosa cell layer. However, immunohistochemical studies suggest that this structure contains the extracellular matrix protein fibronectin (27), apparently produced by granulosa cells (31).
The present immunohistochemical studies on the distribution of factor XIIIA in the chicken follicle (Fig. 6B) demonstrate that this protein is present in the theca externa and possibly in the basement membrane, although this could be demonstrated only with one of the antibodies used (compare Fig. 6A with 6B). Factor XIIIA is completely absent from the theca interna, the granulosa cell layer, and the perivitelline membrane. These results are supported by data obtained from expression studies showing that cells in the theca are the source of the protein. The possibility that factor XIIIA is at least partially recruited from the plasma cannot be excluded at this point. However, it seems rather unlikely, because factor XIIIA in plasma always exists as a heterotetramer complexed to factor XIIIB. Because we could not detect any such complexes in the follicle (Fig. 3B), we suggest that factor XIIIA found in the follicle wall is produced by cells within the follicle, as is the case in the calcifying cartilage discussed above. As shown in Fig. 6B, the localization of XIIIA coincides with that of collagen-1 and fibronectin, both major extracellular matrix components of the theca externa. Because many extracellular matrix proteins, including collagen and fibronectin (32) are substrates for transglutaminases, we assume that factor XIIIA plays a central role in constructing and stabilizing the extracellular matrix of the theca externa. This may provide the mechanical strength necessary for progression through the rapid phase of oocyte growth.
This assumption also applies to the integrity of the postovulatory
follicle, which after loss of its balloon-like tension by expelling the
oocyte, does not disintegrate right away but stays metabolically active
for several more days (2). There is another aspect about factor XIIIA
function in the theca externa: It was recently shown that
transglutaminases and especially factor XIIIA serve as substrates for
cell adhesion (33). Upon inspection of the ultrastructure of the theca
externa (Fig. 6B), it becomes obvious that most of this
tissue is a matrix, with only a few cells scattered in this layer. On
the other hand, a massive presence of factor XIIIA in this tissue
suggests that it serves other functions than catalysis of cross-linking
of matrix proteins. For instance, factor XIIIA may be a true structural
component of the matrix-like fibronectin, serving as an anchor for the
adhesion of the fibroblast-like cells present in this stratum. Finally,
follicular factor XIIIA may also serve the same function as in the
blood. Ovulation of the follicle is started by rupture of the theca
along the stigma, a special region of the follicle that is almost
devoid of blood vessels. However, small capillaries are expected to
rupture upon ovulation and factor XIIIA in the follicular wall might
prevent microbleeding during this event.
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FOOTNOTES |
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* This work was supported by grants from the Austrian Science Foundation (F608 to W. J. S., F606 to J. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Institute of Medical
Biochemistry, Dept. of Molecular Genetics, Biocenter and University of
Vienna, Dr. Bohrgasse 9/2, Vienna A-1030, Austria. Tel.:
43-1-4277-61808; Fax: 43-1-4277-9618; E-mail:
JNIMPF@mol.univie.ac.at.
Published, JBC Papers in Press, August 17, 2000, DOI 10.1074/jbc.M005025200
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ABBREVIATIONS |
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The abbreviations used are: POF, postovulatory follicle; DD, differential display; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; RACE, rapid amplification of cDNA ends; HRP, horseradish peroxidase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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